In modern scientific research, the employment of fluorescent probes has become nearly indispensable as an analytical tool for detecting and quantifying molecular species, including, for example, chemical/biochemical substances.
Such fluorescent probes comprise, in their most basic form, molecules known as fluorophores which, upon the absorption of light, re-radiate this energy as a photon of light that is easily distinguished from scattered excitation light. This process is shown in FIG. 1, schematically representing just such a simple fluorophore (F) compounded with a target molecule (T). As depicted, the fluorophore, upon excitation by absorption of a photon of energy hν1, for instance from a laser, light-emitting diode, etc., is detectable by observing the emission of a fluorescent photon at a lower energy hν2 (the wavelength of the emitted electron is always longer due to loss of some energy in the excited state by reason of molecular motion—e.g., heat). The utility of this most basic structure is limited, however, including the fact that fluorescence is not specific to bound probes.
According to a variant form, shown in FIG. 2, commonly referred to as a dual-labeled FRET (“Förster Resonance Energy Transfer”) probe, there is provided a donor fluorophore (D) which functions to absorb a photon at energy hν1 to produce an excited state donor that, in turn, may emit a photon hν2 as in the previous example. However, the presence of an acceptor fluorophore (A) may, by reason of direct dipole-dipole interactions between the two fluorophores (A and D), result in transfer of the excited state energy from the donor (D) to the acceptor (A) without photon emission. This process results in the quenching of the donor (D) and the formation of an excited state acceptor (A) which may, in turn, emit a photon of a different (longer) wavelength, hν3. The efficiency of this energy transfer depends upon the extent of overlap between the two fluorophores' emission spectra and the relative orientation of their transition dipoles.
Probes utilizing the foregoing structure are capable of a self-quenching functionality. For instance, FRET probes have been developed for nucleic acid hybridization assays wherein the probe hybridizes to itself in the absence of a target molecular species so that the donor and acceptor are in close proximity, thus quenching photon emission. In the presence of the target molecular species, by contrast, the probe preferentially hybridizes this structure to yield an increase in the physical distance between the donor and acceptor to thus permit photon emission and, accordingly, detection of the bound target molecular species.
FRET probes such as described, while conventionally in wide use, suffer from a number of drawbacks including, notably, fluorescent emission from the quencher compound which produces an undesirable background fluorescence.
In an effort to overcome the drawbacks attending both prior art probe variants described above, a third variant form, shown in FIG. 3, has been developed which comprises a fluorophore (F) and a so-called “dark-quencher” (Q). Dark-quenchers may be distinguished from the acceptor fluorophore (A) of FIG. 2 by their characteristic absorption of energy from a light source or another molecule without subsequent emission of a fluorescence photon (the excited state of the dark-quencher returns to the ground state by non-fluorescent decay—e.g., heat). Apart from this distinguishing characteristic, the principle of operation of dark quenchers is quite similar to that of the FRET probes described previously in that, depending upon the distance between the fluorophore (F) and the quencher (Q), the quenching interaction—that is, the absorption of energy by the quencher—may be accomplished through FRET, described above, or via static quenching. And by selectively distancing the fluorophore (F) and quencher (Q), desired fluorescence signaling is triggered. More particularly, when the fluorophore (F) and quencher (Q) are in close proximity (the “quenched state”), a photon hν1 may be absorbed but emission of a photon hν2 cannot occur due to FRET or static quenching. However, at longer distances of separation, such as may be occasioned by a binding or enzymatic cleavage event resulting in the relative separation of the quencher (Q) and fluorophore (F), consequent emission of a fluorescent signal (hν2) is enabled.
Many modifications of the fundamental dark quencher structure described above have been developed. For example, there is provided in the disclosure of Cook et al., International Publication No. WO01/86001 A1, assigned to Biosearch Technologies, Inc., the disclosure of which is incorporated herein by reference in its entirety, dark-quenchers of the exemplary structures of FIG. 4, each of which is characterized by an aromatic donor comprising a N,N-dialkylaniline. Probes comprising such quenchers are advantageous in their characteristic long-wavelength (disclosed to be preferably from about 500 nm to about 700 nm) absorption maxima. However, this particular modification of the dark quencher variant is, in the longest (approximately 673 nm) wavelength quenching version (designated “BHQ-3” in FIG. 4) thereof, characterized by a N-phenylphenazinium moiety which may be susceptible to nucleophilic attack, such as during oligonucleotide deprotection with ammonia and similar reagents, and so creates stability problems when the quencher is conjugated with at least certain carrier molecules comprising the probe.
Accordingly, there continues to exist a need for dark-quenchers, and probes comprising the same, which are at once capable of long-wavelength absorption maxima to accommodate the growing trend towards fluorophores with longer wavelength emission spectra, as well as being characterized by greater stability than that achievable with conventional dark quenchers such as described.